Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells

Hallaq, Haifa; Yang, Zhenjiang; Viswanathan, Prakash C.; Fukuda, Koji; Shen, Wangzhen; Wang, Dao Wen; Wells, K. Sam; Zhou, Jingsong; Yi, Jianxun; Murray, Katherine T.

doi:10.1016/j.cardiores.2006.08.007

Cited by 80 publications

(53 citation statements)

References 40 publications

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“…A human embryonic kidney (HEK)-293 cell line that stably expressed a fusion protein (SCN5A-GFP) with GFP attached to the COOH-terminus of SCN5A (SCN5A, GenBank Accession No. M77235) was generated as previously described (19). In a separate construct, three copies of the HA epitope were inserted into the initial, extracellular portion of the domain I S5-S6 loop of SCN5A (SCN5A-HA), with the construct subcloned into the pCR3 vector as previously described (19).…”

Section: Methodsmentioning

confidence: 99%

“…M77235) was generated as previously described (19). In a separate construct, three copies of the HA epitope were inserted into the initial, extracellular portion of the domain I S5-S6 loop of SCN5A (SCN5A-HA), with the construct subcloned into the pCR3 vector as previously described (19). In addition, we generated a mutant SCN5A-GFP channel in which the conserved PKC site at Ser 1503 was eliminated by a mutation to alanine (SCN5A-S1503A-GFP).…”

Section: Methodsmentioning

confidence: 99%

“…The initial data analysis was performed using LSM510 software. To minimize the effect of light exposure, images were obtained at 5-min intervals unless otherwise stated (19). Average single cell red fluorescence intensity was calculated by dividing the total red channel field intensity (above the background, on scale) by the number of red fluorescent cells.…”

Section: Methodsmentioning

confidence: 99%

“…The combination of 8-chlorophenylthiocAMP (200 M), 3-isobutyl-1-methylxanthine (1 mM), and forskolin (10 M) was used for PKA stimulation (19).…”

Section: Methodsmentioning

confidence: 99%

“…The time-dependent spatial relationship of GFP-tagged SCN5A channels with the Alexa 594-labeled plasma membrane was measured using Metamorph image-analysis software (Universal Imaging, Downington, PA), as previously described (13,19). The dynamic range for both the green (GFP) and red (Alexa 594) channels was maximized to achieve the highest precision and contrast in the image.…”

Section: Methodsmentioning

confidence: 99%

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Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na⁺channels

Hallaq¹,

Wang²,

Kunic³

et al. 2012

American Journal of Physiology-Heart and Circulatory Physiology

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ϩ current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na ϩ channels at the plasma membrane in this effect, but the results are controversial.In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na ϩ currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na ϩ channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca 2ϩ -sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser 1503 or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na ϩ channels in living cells.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The combination of 8-chlorophenylthiocAMP (200 M), 3-isobutyl-1-methylxanthine (1 mM), and forskolin (10 M) was used for PKA stimulation (19).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na⁺channels

Hallaq¹,

Wang²,

Kunic³

et al. 2012

American Journal of Physiology-Heart and Circulatory Physiology

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The late sodium current in heart failure: pathophysiology and clinical relevance

Horváth

Bers

2014

ESC Heart Failure

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Large and growing body of data suggest that an increased late sodium current (I Na,late ) can have a significant pathophysiological role in heart failure and other heart diseases. The first goal of this article is to describe how I Na,late functions under physiological circumstances. The second goal is to show the wide range of cellular mechanisms that can increase I Na,late in cardiac disease, and also to describe how the up-regulated I Na,late contributes to the pathophysiology of heart failure. The final section of the article discusses the possible use of I Na,late -modifying drugs in heart failure, on the basis of experimental and preclinical data.

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Saxitoxin

2014

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Die lähmend wirkende Verbindung (+)‐Saxitoxin (STX), die mit den Roten Tiden der Meere und der paralytischen Schalentiervergiftung assoziiert wird, ist ein potenter Inhibitor der elektrischen Leitung in erregbaren Zellen. Seine tückischen Wirkungen sind eine Folge der Hemmung spannungsgesteuerter Natriumionenkanäle (NaVs), d. h. der Proteine, die für die Initiierung und Fortpflanzung von Aktionspotentialen unerlässlich sind. Ein entscheidender Punkt für den Fortschritt der Ionenkanalforschung und die Identifizierung und Charakterisierung von NaVs war die Verfügbarkeit von STX und des verwandten Guanidiniumderivats Tetrodotoxin. Die Besonderheit von STX zeigt sich in seiner Funktion und Form, weist dieses außerordentlich kompakte Dikation doch mehr Heteroatome als Kohlenstoffzentren auf. Dieser Aufsatz gibt einen Überblick über die Chemie und die chemische Biologie dieses faszinierenden Naturstoffs und zeigt Perspektiven für die gezielte Erforschung der Struktur und Funktion von NaV auf.

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Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells

Abstract: Our results provide direct evidence for PKA-mediated trafficking of cardiac Na(+) channels into the plasma membrane in living, mammalian cells, and they support the existence of multiple intracellular storage pools of channel protein that can be mobilized following a physiologic stimulus.

Cited by 80 publications

References 40 publications

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na⁺channels

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na⁺channels

The late sodium current in heart failure: pathophysiology and clinical relevance

Saxitoxin

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Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells

Abstract: Our results provide direct evidence for PKA-mediated trafficking of cardiac Na(+) channels into the plasma membrane in living, mammalian cells, and they support the existence of multiple intracellular storage pools of channel protein that can be mobilized following a physiologic stimulus.

Cited by 80 publications

References 40 publications

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+channels

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+channels

The late sodium current in heart failure: pathophysiology and clinical relevance

Saxitoxin

Contact Info

Product

Resources

About

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na⁺channels

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na⁺channels